This invention relates to mutagenesis of mycobacteria. More particularly, this invention relates to the generation of insertional mutations in mycobacteria.
Certain mycobacteria represent major pathogens of man and animals. For example, tuberculosis is generally caused in humans by Mycobacterium tuberculosis, and in cattle by Mycobacterium bovis, which may also be transmitted to humans and other animals. Mycobacteria leprae is the causative agent of leprosy. M. tuberculosis and mycobacteria of the avium-intracellulare-scrofulaceum group (MAIS group) represent major opportunistic pathogens of patients with acquired immune deficiency syndrome (AIDS). M. pseudotuberculosis is a major pathogen of cattle.
On the other hand, Bacille Calmette-Guerin, or BCG, an avirulent strain of M. bovis, is widely used in human vaccines, and in particular is used as a live vaccine, which is protective against tuberculosis. BCG is the only childhood vaccine which is currently given at birth, has a very low incidence of adverse effects, and can be used repeatedly in an individual (e.g., in multiple forms). In addition, BCG and other mycobacteria (e.g., M. smegmatis), employed in vaccines, have adjuvant properties among the best currently known and, therefore, stimulate a recipient""s immune system to respond to antigens with great effectiveness.
It has been suggested by Jacobs, et al, Nature, Vol. 327, No. 6122, pgs. 532-535 (Jun. 11, 1987) that BCG could be used as a host for the construction of recombinant vaccines. In other words, it was suggested to take an existing vaccine (in this case against tuberculosis) and expand its protective repertoire through the introduction of one or more genes from other pathogens. Because BCG vaccines are administered as live bacteria, it is essential that any foreign antigens, polypeptides, or proteins expressed by the bacteria are not lost from the bacteria subsequent to vaccination.
Transformation, the process whereby naked DNA is introduced into bacterial cells, has been carried out successfully in mycobacteria. Jacobs, et al (1987), hereinabove cited, have described transformation of mycobacteria through chemical methods, and Snapper, et al. PNAS, Vol. 85, pgs. 6987-6991 (September 1988) have described transformation of mycobacteria by electroporation. Electroporation can give from 105 to 106 transformants per xcexcg of plasmid DNA and such plasmid DNA""s may carry genes for resistance to antibiotic markers such as kanamycin (Snapper, et al 1988) to allow for selection of transformed cells from non-transformed cells.
Jacobs, et al (1987) and Snapper, et al (1988) have also described the use of cloning vehicles, such as plasmids and bacteriophages, for carrying genes of interest into mycobacteria.
Combination of the above-mentioned techniques, along with standard tools of molecular cloning (e.g., use of restriction enzymes, etc.) allows the cloning of genes of interest into vectors and introduction of such genes into mycobacteria. To express these genes, it is important to have available signals for gene expression, in particular, transcription promoter elements. Such promoter elements have been isolated from mycobacterial heat shock genes, and have been used to express foreign antigens in mycobacteria.
Molecular genetics of mycobacteria, however has only recently begun to be developed, in part because mycobacteria present formidable obstacles to genetic study in that mycobacteria, in general, clump in culture and grow very slowly. The direct selection of mutants by employing transposons (also known as random insertional mutagenesis) has been a useful approach to the mutational analysis of microbial pathogenesis (Isberg, et al., Curr. Top. Microbiol. Immunol., Vol. 118, pgs. 1-11 (1985); Taylor, et al., J. Bacteriol, Vol. 171, pgs. 1870-1878 (1989); Fields, et al., Science, Vol. 243, pgs. 1059-1061 (1989); Bernardini, et al., Proc. Nat. Acad. Sci., Vol. 86, pgs. 3867-3871 (1989)); such selection of mutants, however, had not been described in mycobacteria.
Objects of the present invention include the generation of mutations in mycobacteria and/or the introduction of heterologous genes into mycobacteria; in particular, the generation of mutations of mycobacteria employed in vaccines, such as BCG, as well as the generation of mutations in pathogenic mycobacteria, such as M. tuberculosis or M. leprae, whereby such mutations make the mycobacteria non-pathogenic. Heterologous genes which may be introduced into the mycobacteria include, but are not limited to, genes for protective antigen(s) for a variety of pathogens, and/or for other therapeutic agents.